体内和体外高效表达大肠杆菌水通道蛋白Z
|
摘要
大肠杆菌水通道蛋白Z(AqpZ)水选择专一性强、渗透性高、结构稳定,所以AqpZ在仿生废水回收以及海水淡化等领域具有潜在的重要应用价值。但是由于AqpZ强烈疏水性会导致细胞毒性,使得在传统大肠杆菌体系表达量仅仅只有2.5 mg/l,很难满足制备基于水通道蛋白的水过滤装置的需求。
本工作的目的是运用融合表达策略和无细胞体系表达策略,高效制备功能性AqpZ,为制备新型水过滤装置奠定坚实的基础,并发展一个具有普遍意义的膜蛋白高效表达技术。
首先采用融合表达策略,即在目标膜蛋白的上游引入亲水性分子伴侣,构建带有不同融合标签的体内表达载体。发现pMAL-P2-AqpZ对宿主细胞生长的影响最小,融合蛋白的表达水平最高,进一步优化诱导条件(包括诱导时间、培养温度、IPTG浓度、诱导后培养时间),表达量可高达200 mg/l。
由于AqpZ疏水性会导致细胞毒性,随后采用没有生长要求的无细胞蛋白质合成体系。构建的一系列带有不同N端序列的无细胞体系表达载体中,pIVEX2.4c-AqpZ为最佳表达载体。由于无细胞体系缺少膜蛋白正确折叠所需要的疏水环境,AqpZ都是以沉淀的形式表达。首先采用去污剂重悬的模式来制备可溶性AqpZ,然而采用的10余种去污剂均不能有效溶解AqpZ沉淀。考虑到无细胞体系的开放性特点,采用直接添加去污剂的模式,发现Brij78为最优去污剂,在112xCMC最佳工作浓度下,可溶表达水平约为530 mg/l。此外,还研究了直接添加脂质体的模式,在实验最高脂质体浓度(5 mg/ml)下,整合到磷脂膜的AqpZ表达水平达到470 mg/l。
在无细胞体系表达AqpZ时发现,mRNA翻译起始区形成二级结构会抑制翻译的有效启动,使得不带有N端标签的AqpZ表达量较低,所以开展了无细胞体系高效表达AqpZ新策略方面的研究。首先采用双顺反子策略,虽然构建的双顺反子质粒能够有效提高AqpZ在大肠杆菌体系的表达水平,但这些质粒并不能在无细胞体系实现高效表达。随后采用信号肽策略,构建带有不同信号肽序列的AqpZ体外表达载体,目标蛋白在无细胞体系的表达量提高了5倍以上;且通过添加去污剂或者脂质体可以激活无细胞抽提物中的信号肽酶活性,实现信号肽序列的原位切除,说明信号肽序列可以作为促进蛋白翻译的序列元件而发展一个具有普遍意义的、与目的蛋白DNA序列无关的膜蛋白高效表达新策略。
Aquaporin Z (AqpZ), the water channel protein from Escherichia coli, is an orthodox aquaporin with high water selectivity and high permeability, making AqpZ-based water filter a promising tool for water reuse and seawater desalination. However, the development of this novel bio-filter is hampered by the availability of sufficient amount of water channel proteins since the overexpression of integral membrane proteins may exhibit toxicity towards the host due to the highly hydrophobic nature.
The aim of this work is to synthesize functional AqpZ at high levels either by fusion expression strategy or cell-free expression system, which lays a solid foundation to construct the novel aquaporin-based water filter, and also promises a universal platform for efficient production of membrane proteins.
Firstly, fusion expression was chosen to improve the expression level of AqpZ. By fusion with several hydrophilic partners, the yield of AqpZ was significantly improved, and MBP was determined to be the most efficient fusion partner to increase the expression level. After systematically investigating the effects of different induction conditions, such as temperature, induction-timing, IPTG concentration and post-induction duration, high productivity of MBP-AqpZ (200 mg/l) was achieved.
Because the overexpression of membrane proteins would lead to cell toxicity, no growth-required cell-free protein synthesis (CFPS) system was employed as an alternative to synthesize AqpZ. Several in vitro expression vectors with different N-terminal sequence were constructed, and pIVEX2.4c-AqpZ was determined to be the most efficient vector in cell-free system. However, due to the lack of hydrophobic environment essential for membrane protein folding, AqpZ was aggregated as precipitates in a standard CFPS. In order to produce soluble AqpZ, detergent resolubilization was investigated first. However, all the tested detergents in this work failed to resolubilize the AqpZ precipitates efficiently. Therefore, detergents were supplemented directly instead, taking advantage of the open nature of CFPS. By systematically screening and optimizing of detergent structure and concentration, the highest soluble expression (530 mg/1) was achieved by Brij78 at a final concentration of 112xCMC. In addition, DOPC liposome supplementation was also investigated, and 470 mg/1 membrane-associated AqpZ was achieved at the highest DOPC concentration tested (5 mg/ml).
Furthermore, the formation of mRNA secondary structure was found to inhibit translation initiation, which limited the expression level of native AqpZ in cell-free system. Therefore, research work on two-cistronic plasmid strategy and leader peptide strategy were undertaken. Although the constructed two-cistronic plasmids could improve the expression level of AqpZ in vivo, these plasmids didn't work well in cell-free system. On the contrary, by fusion with many naturally occurring leader peptides, the expression level of AqpZ was enhanced by as much as 5 times through avoiding the formation of secondary structure. In addition, the leader peptide could be cleaved in situ in cell-free system supplemented with detergent or liposome, through the activation of leader peptidase. These results indicated that leader peptide sequence could be used as a downstream box to stimulate recombinant protein synthesis in cell-free system, promising a universal platform for efficient production of membrane proteins, regardless of the nature of target DNA sequence.
本工作的目的是运用融合表达策略和无细胞体系表达策略,高效制备功能性AqpZ,为制备新型水过滤装置奠定坚实的基础,并发展一个具有普遍意义的膜蛋白高效表达技术。
首先采用融合表达策略,即在目标膜蛋白的上游引入亲水性分子伴侣,构建带有不同融合标签的体内表达载体。发现pMAL-P2-AqpZ对宿主细胞生长的影响最小,融合蛋白的表达水平最高,进一步优化诱导条件(包括诱导时间、培养温度、IPTG浓度、诱导后培养时间),表达量可高达200 mg/l。
由于AqpZ疏水性会导致细胞毒性,随后采用没有生长要求的无细胞蛋白质合成体系。构建的一系列带有不同N端序列的无细胞体系表达载体中,pIVEX2.4c-AqpZ为最佳表达载体。由于无细胞体系缺少膜蛋白正确折叠所需要的疏水环境,AqpZ都是以沉淀的形式表达。首先采用去污剂重悬的模式来制备可溶性AqpZ,然而采用的10余种去污剂均不能有效溶解AqpZ沉淀。考虑到无细胞体系的开放性特点,采用直接添加去污剂的模式,发现Brij78为最优去污剂,在112xCMC最佳工作浓度下,可溶表达水平约为530 mg/l。此外,还研究了直接添加脂质体的模式,在实验最高脂质体浓度(5 mg/ml)下,整合到磷脂膜的AqpZ表达水平达到470 mg/l。
在无细胞体系表达AqpZ时发现,mRNA翻译起始区形成二级结构会抑制翻译的有效启动,使得不带有N端标签的AqpZ表达量较低,所以开展了无细胞体系高效表达AqpZ新策略方面的研究。首先采用双顺反子策略,虽然构建的双顺反子质粒能够有效提高AqpZ在大肠杆菌体系的表达水平,但这些质粒并不能在无细胞体系实现高效表达。随后采用信号肽策略,构建带有不同信号肽序列的AqpZ体外表达载体,目标蛋白在无细胞体系的表达量提高了5倍以上;且通过添加去污剂或者脂质体可以激活无细胞抽提物中的信号肽酶活性,实现信号肽序列的原位切除,说明信号肽序列可以作为促进蛋白翻译的序列元件而发展一个具有普遍意义的、与目的蛋白DNA序列无关的膜蛋白高效表达新策略。
Aquaporin Z (AqpZ), the water channel protein from Escherichia coli, is an orthodox aquaporin with high water selectivity and high permeability, making AqpZ-based water filter a promising tool for water reuse and seawater desalination. However, the development of this novel bio-filter is hampered by the availability of sufficient amount of water channel proteins since the overexpression of integral membrane proteins may exhibit toxicity towards the host due to the highly hydrophobic nature.
The aim of this work is to synthesize functional AqpZ at high levels either by fusion expression strategy or cell-free expression system, which lays a solid foundation to construct the novel aquaporin-based water filter, and also promises a universal platform for efficient production of membrane proteins.
Firstly, fusion expression was chosen to improve the expression level of AqpZ. By fusion with several hydrophilic partners, the yield of AqpZ was significantly improved, and MBP was determined to be the most efficient fusion partner to increase the expression level. After systematically investigating the effects of different induction conditions, such as temperature, induction-timing, IPTG concentration and post-induction duration, high productivity of MBP-AqpZ (200 mg/l) was achieved.
Because the overexpression of membrane proteins would lead to cell toxicity, no growth-required cell-free protein synthesis (CFPS) system was employed as an alternative to synthesize AqpZ. Several in vitro expression vectors with different N-terminal sequence were constructed, and pIVEX2.4c-AqpZ was determined to be the most efficient vector in cell-free system. However, due to the lack of hydrophobic environment essential for membrane protein folding, AqpZ was aggregated as precipitates in a standard CFPS. In order to produce soluble AqpZ, detergent resolubilization was investigated first. However, all the tested detergents in this work failed to resolubilize the AqpZ precipitates efficiently. Therefore, detergents were supplemented directly instead, taking advantage of the open nature of CFPS. By systematically screening and optimizing of detergent structure and concentration, the highest soluble expression (530 mg/1) was achieved by Brij78 at a final concentration of 112xCMC. In addition, DOPC liposome supplementation was also investigated, and 470 mg/1 membrane-associated AqpZ was achieved at the highest DOPC concentration tested (5 mg/ml).
Furthermore, the formation of mRNA secondary structure was found to inhibit translation initiation, which limited the expression level of native AqpZ in cell-free system. Therefore, research work on two-cistronic plasmid strategy and leader peptide strategy were undertaken. Although the constructed two-cistronic plasmids could improve the expression level of AqpZ in vivo, these plasmids didn't work well in cell-free system. On the contrary, by fusion with many naturally occurring leader peptides, the expression level of AqpZ was enhanced by as much as 5 times through avoiding the formation of secondary structure. In addition, the leader peptide could be cleaved in situ in cell-free system supplemented with detergent or liposome, through the activation of leader peptidase. These results indicated that leader peptide sequence could be used as a downstream box to stimulate recombinant protein synthesis in cell-free system, promising a universal platform for efficient production of membrane proteins, regardless of the nature of target DNA sequence.
引文
[1]王镜岩,朱圣庚,徐长法主编。生物化学(第三版,上册)。北京:高等教育出版社,2002,P589-595
[2]Singer SJ,Nicolson GL.The fluid mosaic model of the structure of cell membranes.Science,1972,175:720-731
[3]Rothman JE,Lenard J.Membrane asymmetry.Science,1997,195:743-753
[4]隋森芳编著。膜分子生物学。北京:高等教育出版社,2003,P1-5
[5]Bowie JU.Solving the membrane protein folding problem.Nature,2005,438:581-589
[6]Brunecky R,Lee S,Rzepecki PW,Overduin M,Prestwich GD,Kutateladze AG,Kutateladze TG.Investigation of the binding geometry of a peripheral membrane protein.Biochemistry,2005,44(49):16064-16071
[7]Cho W,Stahelin RV.Membrane-protein interactions in cell signaling and membrane trafficking.Annu Rev Biophys Biomol Struct,2005,34:119-151
[8]Gao FP,Cross TA.Recent developments in membrane-protein structural genomics.Genome Biol,2005,6(13):244
[9]Dailey MM,Hait C,Holt PA,Maguire JM,Meier JB,Miller MC,Petraccone L,Trent JO.Structure-based drug design:from nucleic acid to membrane protein targets.Exp Mol Pathol,2009,86(3):141-50
[10]Faller LD.Mechanistic studies of sodium pump.Arch Biochem Biophys,2008,476(1):12-21
[11]Staudinger JL,Lichti K.Cell signaling and nuclear receptors:new opportunities for molecular pharmaceuticals in liver disease.Mol Pharm,2008,5(1):17-34
[12]Wang K,Wong YH.G protein signaling controls the differentiation of multiple cell lineages.Biofactors,2009,35(3):232-8.
[13]Williams C,Hill SJ.GPCR signaling:understanding the pathway to successful drug discovery.Methods Mol Biol,2009,552:39-50
[14] Membrane Protein of Known 3D Structure, [December 2009],http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
[15] Protein Data Bank, [December 2009], http://www.rcsb.org/pdb/home/home.do
[16] Grisshammer R, Tate CG. Overexpression of integral membrane proteins for structural studies. Q Rev Biophys, 1995, 28: 315-422
[17] Ress DC, Chang G, Spencer RH. Crystallographic analyses of ion chennels:Lessons and challenges. J Biol Chem, 2000, 275: 32399-32402
[18] Haines TH. Water transport across biological membranes. FEBS Lett, 1994, 346:115-122
[19] Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science, 1992, 256: 385-387
[20] Agre P, Brown D, Nielsen S. Aquaporin water channels: unanswered questions and unresolved controversies. Curr Opin Cell Biol, 1995, 7: 472-483
[21] Agre P. Aquaporin water channels (Nobel Lecture). Bioscience Rep, 2004, 24(3):127-172
[22] Verkman AS. More than just water channels: unexpected cellular roles of aquaporins. J Cell Sci, 2005, 118(15): 3225-3232
[23] Ishibashi K, Hara S, Kondo S. Aquaporin water channels in mammals. Clin Exp Nephrol, 2009, 13(2): 107-17
[24] Schrier RW. Aquaporin-related disorders of water homeostasis. Drug News Perspect, 2007, 20(7): 447-53
[25] Chepelinsky AB. Structural function of MIP/aquaporin 0 in the eye lens; genetic defects lead to congenital inherited cataracts. Handb Exp Pharmacol, 2009, 190:265-97
[26] Oshio K, Song Y, Verkman AS, Manley GT. Aquaporin-1 deletion reduces osmotic water permeability and cerebrospinal fluid production. Acta Neurochir Suppl,2003, 86: 525-528.
[27] Nishimura H, Yang Y, Lau K, Kuykindoll RJ, Fan Z, Yamaguchi K, Yamamoto T.Aquaporin-2 water channel in developing quail kidney: possible role in programming adult fluid homeostasis. Am J Physiol Regul Integr Comp Physiol, 2007, 293(5): R2147-R2158
[28] Benarroch EE. Aquaporin-4, homeostasis, and neurologic disease. Neurology, 2007, 69(24): 2266-2268
[29] Galamita G, Bishai WR, Preston GM, Guggino WB, Agre P. Molecular cloning and characterization of AqpZ, a water channel from E. coli. J Biol Chem, 1995, 49:29063-29066
[30] Calamita G, Kempf B, Rudd KE, Bonhivers M, Kneip S, Bishai WR, Bremerd E,Agre P. The aquaporin-Z water channel gene of Escherichia coli: Structure,organization and phylogeny. Biol Cell, 1997, 89: 321-329
[31] Calamita G. The Escherichia coli aquaporin-Z water channel. Mol Microbiol,2000, 37(2): 254-262
[32] Borgnia MJ, Kozono D, Calamita G, Maloney PC, Agre P. Functional Reconstitution and Characterization of AqpZ, the E. coli Water Channel Protein. J Mol Biol, 1999,291: 1169-1179
[33] Daniels BV, Jiang JS, Fu D. Crystallization and preliminary crystallograpgic analysis of the Escherichia coli water channel AqpZ. Acta Cryst D, 2004, 60: 561-563
[34] Jiang JS, Daniels BV, Fu D. Crystal structure of AqpZ tetramer reveals two distinct Arg-189 conformations associated with water permeation through the narrowest constriction of the water-conducting channel. J Biol Chem, 2006, 281(1):454-460
[35] Ringler P, Borgnia MJ, Stahlberg H, Maloney PC, Agre P, Engel A. Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography. J Mol Biol, 1999,291: 1181-1190
[36] Scheuring S, Ringler P, Borgnia M, Stahlberg H, Muller DJ, Agre P, Engel A.High resolution AFM topographs of the Escherichia coli water channel aquaporin Z.EMBO J, 1999, 18(18): 4981-4987
[37] Savage DF, Egea PF, Robles-Colmenares Y, O'Connell JD, Stroud RM. Architecture and selectivity in aquaporins: 2.5 a X-ray structure of aquaporin Z. PLoS Biol, 2003, 1(3): E72
[38] Jensen M, Mouritsen OG. Single-channel water permeabilities of Escherichia coli aquaporins: AqpZ and GlpF. Biophys J, 2006, 90: 2270-2284
[39] Phongphanphanee S, Yoshida N, Hirata F. The statistical-mechanics study for the distribution of water molecules in aquaporin. Chem Phys Lett, 2007,449: 196-201
[40] Wang Y, Schulten K, Tajkhorshid E. What makes an aquaporin a glycerol channel?A comparative study of AqpZ and GlpF. Structure, 2005,13: 1107-1118
[41] Hashido M, Ikeguchi M, Kidera A. Comparative simulations of aquaporin family:AQP1, AQPZ, AQP0 and GlpF. FEBS Lett, 2005, 579: 5549-5552
[42] Pohl P, Saparov SM, Borgnia MJ, Agre P. Highly selective water channel activity measured by voltage clamp: Analysis of planar lipid bilayers reconstituted with purified AqpZ. Proc Natl Acad Sci USA, 2001, 98(17): 9624-9625
[43] Fujiyoshi Y, Mitsuoka K, de Groot BL, Philippsen A, Grubmüller H, Agre P,Engel A. Structure and function of water channels. Curr Opin Struct Biol, 2002, 12(4):509-15
[44] Verkman AS, Mitra AK. Structure and function of aquaporin water channels. Am J Physiol Renal Physiol, 2000, 278(1): F13-F28
[45] Kumar M, Grzelakowski M, Zilles J, Clark M, Meier W. Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z. Proc Natl Acad Sci USA, 2007, 104(52): 20719-20724
[46] Swartz JR. Developing cell-free biology for industrial applications. J Ind Microbiol Biotechnol, 2006, 33: 476-485
[47] Haddoub R, Rutzler M, Robin A, Flitsch SL. Design, synthesis and assaying of potential aquaporin inhibitors. Handb Exp Pharmacol, 2009, 190: 385-402.
[48] Castle NA. Aquaporins as targets for drug discovery. Drug Discov Today, 2005,10(7): 485-93
[49] Yasui M. Molecular mechanisms and drug development in aquaporin water channel diseases: structure and function of aquaporins. J Pharmacol Sci, 2004, 96(3):260-3
[50] Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A,Nielsen S. Aquaporin water channels-from atomic structure to clinical medicine. J Physiol, 2002, 542(1): 3-16
[51] Wang DN, Safferling M, Lemieux MJ, Griffith H, Chen Y, Li XD. Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies. Biochim Biophys Acta, 2003, 1610: 23-36
[52] Tate CG. Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett, 2001, 504(3): 94-98
[53] Wuu JJ, Swartz JR. High yield cell-free production of integral membrane proteins without refolding or detergents. Biochim Biophys Acta, 2008, 1778:1237-1250
[54] Miroux B, Walker JE. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol, 1996,260: 289-298
[55] Arechaga I, Miroux B, Karrasch S, Huijbregts R, Kruijff BD, Runswick MJ,Walker JE. Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F_1F_0 ATP synthase.FEBS Lett, 2000, 482: 215-219
[56] Mohanty AK, Wiener MC. Membrane protein expression and production: effects of polyhistidine tag length and position. Protein Expre Purif, 2004, 33: 311-325
[57] Link JA, Skretas G, Strauch EM, Chari NS, Georgiou G. Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli. Protein Sci, 2008, 17: 1857-1863
[58] Skretas G, Georgiou G. Genetic analysis of G protein-coupled receptor expression in Escherichia coli: Inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor. Biotechnol Bioeng, 2009, 102(2): 357-367
[59] Kiefer H, Vogel R, Maier K. Bacterial expression of G-protein-coupled receptors:prediction of expression levels from sequence. Receptors Channels, 2000, 7: 109-119
[60] Kiefer H, Krieger J, Olszewski JD, Von Heijne G, Prestwich GD, Breer H.Expression of an olfactory receptor in Escherichia coli: purification, reconstitution,and ligand binding. Biochemistry, 1996, 35: 16077-16084
[61] Korepanova A, Moore JD, Nguyen HB, Hua Y, Cross TA, Gao F. Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein. Protein Expr Purif, 2007, 53: 24-30
[62] Chen GQ, Gouaux JE. Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: Efficient regeneration of the fusion protein and selective cleavage with trypsin. Protein Sci, 1996,5:456-467
[63] Pompejus M, Friedrich K, Teufel M, Fritz HJ. High-yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli. Eur J Biochem, 1993,211:27-35
[64] Yao Q, Bevan JL, Weaver RF, Bigelow DJ. Purification of porcine phospholamban expressed in Escherichia coli. Protein Expres Purif, 1996, 8: 463—468
[65] Buck B, Zamoon J, Kirby TL, DeSilva TM, Karim C, Thomas D, Veglia G.Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies. Protein Expres Purif, 2003, 30: 253-261
[66] Arkin IT, Adams PD, MacKenzie KR, Lemmon MA, Brunger AT, Engelman DM.Structural organization of the pentameric transmembrane alpha-helices of phospholamban, a cardiac ion channel. EMBO J, 1994, 13: 4757-4764
[67] Grisshammer R, Duckworth R, Henderson R. Expression of a rat neurotensin receptor in Escherichia coli. Biochem J, 1993, 295: 571-576
[68] Tucker J, Grisshammer R. Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem J, 1996, 317: 891-899
[69] Stanasila L, Massotte D, Kieffer BL, Pattus F. Expression of δ, μ and κ human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins. Eur J Biochem, 1999, 260: 430-438
[70] Lemmon MA, Flanagan JM, Hunt JF, Adair BD, Bormann BJ, Dempsey CE,Engelman DM. Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices. J Biol Chem, 1992, 267: 7683-7689
[71] Ramachandran S, Lu HL, Prabhu U, Ruoho AE. Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli. Protein Expr Purif, 2007, 51: 283-292
[72] Weiss HM, Grisshammer R. Purification and characterization of the human adenosine A_(2a) receptor functionally expressed in Escherichia coli. Eur J Biochem,2002, 269: 82-92
[73] Roosild TP, Greenwald J, Vega M, Castronovo S, Riek R, Choe S. NMR structure of Mistic, a membrane-integrating protein for membrane protein expression.Science, 2005, 307: 1317-1321
[74] Kulothungan SR, Das M, Johnson M, Ganesh C, Varadarajan R. Effect of crowding agents, signal peptide, and chaperone SecB on the folding and aggregation of E. coli maltose binding protein. Langmuir, 2009,25(12): 6637-48.
[75] Katzen F, Chang G, Kudlicki W. The past, present and future of cell-free protein synthesis. Trends Biotechol, 2005, 23: 150-156
[76] Klammt C, Schwarz D, Lohr F, Schneider B, Dotsch V, Bernhard F. Cell-free expression as an emerging technique for the large scale production of integral membrane protein. FEBS J, 2006, 273: 4141-4153
[77] Schwarz D, Klammt C, Koglin A, Lohr F, Schneider B, Dotsch V, Bernhard F.Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies. Methods, 2007, 41: 355-369
[78] Liguori L, Marques B, Villegas-Mendez A, Rothe R, Lenormand JL. Production of membrane proteins using cell-free expression systems. Expert Rev Proteomics,2007, 4(1): 79-90
[79] Endo Y, Sawasaki T. Cell-free expression systems for eukaryotic protein production. Curr Opin Biotechnol, 2006, 17: 373-380
[80] Yin G, Swartz JR. Enhancing multiple disulfide bonded protein folding in a cell-free system. Biotechnol Bioeng, 2004, 86:188-195
[81] Goerke AR, Swartz JR. Development of cell-free protein synthesis platforms for disulfide bonded proteins. Biotechnol Bioeng, 2008, 99: 351-367
[82] Klammt C, Lohr F, Schafer B, Haase W, Dotsch V, Ruterjans GC, Bernhard F.High level cell-free expression and specific labeling of integral membrane proteins.Eur J Biochem, 2004, 271:568-580
[83] Goerke AR, Swartz JR. High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids. Biotechnol Bioeng, 2009, 102(2):400-416
[84] Rungpragayphan S, Nakano H, Yamane T. PCR-linked in vitro expression: a novel system for high-throughput construction and screening of protein libraries.FEBS Lett, 2003, 540: 147-150
[85] Keller T, Schwarz D, Bernhard F, Dotsch V, Hunte C, Gorboulev V, Koepsell H.Cell free expression and functional reconstitution of eukaryotic drug transporters.Biochemistry, 2008,47: 4552-4564
[86] Shimada Y, Wang ZY, Mochizuki Y, Kobayashi M, Nozawa T. Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell free synthesis system. Biosci Biotechnol Biochem, 2004,68(9): 1942-1948
[87] Klammt C, Schwarz D, Fendler K, Haase W, Dotsch V, Bernhard F. Evaluation of detergents for the soluble expression of a-helical and β-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system. FEBS J, 2005,272: 6024-6038
[88] Elbaz Y, Steiner-Mordoch S, Danieli T, Schuldiner S. In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state. Proc Natl Acad Sci USA, 2004, 101: 1519-1524
[89] Berrier C, Park KH, Abes S, Bibonne A, Betton JM, Ghazi A. Cell free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. Biochemistry, 2004, 43: 12585-12591
[90] Klammt C, Schwarz D, Eifler N, Engel A, Piehler J, Haase W, Hahn S, Dotsch V,Bernhard F. Cell-free production of G protein-coupled receptors for functional and structural studies. J Struct Biol, 2007, 158: 482-493
[91] Gourdon P, Alfredsson A, Pedersen A, Malmerberg E, Nyblom M, Widell M,Berntsson R, Pinhassi J, Braiman M, Hansson O, Bonander N, Karlsson G, Neutze R. Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump. Protein Expr Purif, 2008, 58: 103-113
[92] Martin M, Albanesi D, Alzari M, Mendoza D. Functional in vitro assembly of the integral membrane bacterial thermosensor DesK. Protein Expr Purif, 2009, 66: 39-45
[93] Ishihara G, Goto M, Saeki M, Ito K, Hori T, Kigawa T, Shirouzu M, Yokoyama S.Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin fusion vectors. Protein Expr Purif, 2005,41: 27-37
[94] Liguori L, Marques B, Lenormand JL. A bacterial cell-free expression system to produce membrane proteins and proteoliposomes: from cDNA to functional assay.Curr Protoc Protein Sci, 2008, 5.22.1-5.22.30
[95] Liguori Lavini, Blesneac I, Madern D, Vivaudou M, Lenormand JL. Single-step production of functional OEP24 proteoliposomes. Protein Expr Purif, 2010, 69:106-111
[96] Marques B, Liguori L, Paclet M-H, Villegas-Mendez A, Rothe R, Morel F,Lenormand JL. Liposome-Mediated Cellular Delivery of Active gp91~(phox). PLoS ONE,2007,2(9): e856
[97] Shim JW, Yang MM, Gu LQ. In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv. FEBS Lett, 2007,581: 1027-1034
[98] van Dalena A, van der Laanb M, Driessenb A, Killiana JA, de Kruijff B.Components required for membrane assembly of newly synthesized K~+ channel KcsA.FEBS Lett, 2002, 511: 51-58
[99] Kalmbach R, Chizhov I, Schumacher MC, Friedrich T, Bamberg E, Engelhard M.Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. J Mol Biol, 2007, 371: 639-648
[100] Nagamori S, Vazquez-Ibar JL, Weinglass AB, Kaback HR. In vitro synthesis of lactose permease to probe the mechanism of membrane insertion and folding. J Biol Chem, 2003, 278: 14820-14826
[101] Liguori L, Marques B, Villegas-Mendez A, Rothe R, Lenormand JL.Liposomes-mediated delivery of pro-apoptotic therapeutic membrane proteins. J Control Release, 2008, 126: 217-227
[102] Park KH, Berrier C, Lebaupain F, Pucci B, Popot JL, Ghazi A, Zito F.Flurinated and hemiflurinated surfactants as alternatives to detergents for membrane protein cell-free synthesis.Biochem J,2007,403:183-187
[103]Schnaitman CA.Protein Composition of the cell wall and cytoplasmic membrane of Escherichia coli.J Bacteriol,1970,104(2):890-901
[104]Peker B,Wuu JJ,Swartz JR.Affinity purification of lipid vesicles.Biotechnol Prog,2004,20:262-268
[105]Sambrook J,Fritsch E F,Maniatis T.分子克隆实验指南(第三版),北京:科学出版社,2002,P26-99
[106]Coligan JE,Dunn BM,Speicher DW,Wingfield PT.精编蛋白质科学实验指南,北京:科学出版社,2007,P291-334
[107]LaVallie ER,DiBlasio EA,Kovacic S,Grant KL,Schendel PF,McCoy JM.A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E.coli cytoplasm.Biotechnology,1993,11:187-193
[108]Zhang Y,Olsen DR,Nguyen KB,Olson PS,Rhodes ET,Mascarenhas D.Expression of eukaryotic proteins in soluble form in Escherichia coli.Protein Expres Purif,1998,12:159-165
[109]Nygren PA,Stahl S,Uhlen M.Engineering proteins to facilitate bioprocessing.Trends Biotechnol,1994,12:184-188
[110]Samuelsson E,Moks T,Nilsson B,Uhlen M.Enhanced in vitro refolding of insulin-like growth factor I using a solubilizing fusion partner.Biochemistry,1994,33:4207-4211
[111]Power RF,Conneely OM,McDonnell DP,Clark JH,Butt TR,Schrader WT,O'Malley BW.High level expression of a truncated chicken progesterone receptor in Escherichia coli.J Biol Chem,1990,265:1419-1424
[112]Pryor KD,Leiting B.High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding protein double-affinity fusion system.Protein Expres Purif,1997,10:309-319
[113]Peng L,Xu ZN,Fang XM,Wang F,Cen PL.High-level expression of soluble human Beta-Defensin-2 in E.coli.Process Biochem,2004,39:2199-2205
[114]Xu ZN,Zhong ZX,Huang L,Peng L,Wang F,Cen PL.High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression. Appl Microbiol Biotechnol, 2006, 72: 471-479
[115] Zhong ZX, Xu ZN, Peng L, Huang L, Fang XM, Cen PL. Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli.Appl Microbiol Biotechnol, 2006, 71(5): 661-667
[116] Kozono D, Ding X, Iwasaki I, Meng X, Kamagata Y, Agre P, Kitagawa Y.Functional expression and characterization of an archaeal aquaporin: AqpM from Methanothermobactermarburgensis. J Biol Chem, 2003,278(12): 10649-10656
[117] Hiroaki Y, Tani K, Kamegawa A, Gyobu N, Nishikawa K, Suzuki H, Walz T,Sasaki S, Mitsuoka K, Kimura K, Mizoguchi A, Fujiyoshi Y. Implications of the aquaporin-4 structure on array formation and cell adhesion. J Mol Biol, 2006, 355(4):628-639
[118] Karlsson M, Fotiadis D, Sjovall S, Johansson I, Hedfalk K, Engel A, Kjellbom P.Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris. FEBS Lett, 2003, 537(1-3): 68-72
[119] Hall MN, Gabay J, Debarbouille M, Schwartz M. A role for mRNA secondary structure in the control of translation initiation. Nature, 1982, 295: 616-618
[120] Brodsky LI, Ivanov VV, Kalaydzidis YL, Leontovich AM, Nikolaev VK,Feranchuk SI, Drachev VA. GeneBee-NET: Internet-based server for analyzing biopolymers structure. Biochemistry, 1995, 60: 923-928
[121] DeGrip WJ. Thermal stability of rhodopsin and opsin in some novel detergents.Methods Enzymol, 1982, 81: 256-265
[122] Odahara T. Stability and solubility of integral membrane proteins from photosynthetic bacteria solubilized in different detergents. Biochim Biophys Acta,2004, 1660: 80-92
[123] Ganoza MC, Kofoid EC, Marliere P, Louis BG. Potential secondary structure at translation-initiation sites. Nucleic Acids Res, 1987, 15(1): 345-360
[124] de Smit MH, van Duin J. Secondary structure of the ribosome binding site determines translational efficiency: A quantitative analysis. Proc Natl Acad Sci USA,1990, 87: 7668-7672
[125] Kimura S, Iyanari T. High-level expression of porcine liver cytochrome P-450 reductase catalytic domain in Escherichia coli by modulating the predicted local secondary structure of mRNA. J Biochem, 2003, 134: 403-413
[126] Son JM, Ahn JH, Hwang MY, Park CG, Choi CY, Kim DM. Enhancing the efficiency of cell-free protein synthesis through the polymerase-chain-reaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues. Anal Biochem, 2006, 351: 187-192
[127] Kimura S, Umemura T, Iyanagi T. Two-cistronic expression plasmids for high-level gene expression in Escherichia coli preventing translational initiation inhibition caused by the intramolecular local secondary structure of mRNA. J Biochem, 2005, 137: 523-533
[128] Tschantz WR, Paetze M, Cao GQ, Suciu D, Inouye M, Dalbey RE. Characterization of a soluble, catalytically active form of Escherichia coli leader peptidase: requirement of detergent or phospholipid for optimal activity. Biochemistry 1995,34:3935-3941
[129] Choo KH, Tan TW, Ranganathan S. SPdb - a signal peptide database. BMC Bioinformatics, 2005, 6: 249
[130] Katzen F, Peterson TC, Kudlicki W. Membrane protein expression: no cells required. Trends Biotechnol, 2009, 27(8): 455-460
[131] Cappuccio JA, Blanchette CD, Sulchek TA, Arroyo ES, Kralj JM, Hinz AK,Kuhn EA, Chromy BA, Segelke BW, Rothschild KJ, Fletcher JE, Katzen F, Peterson TC, Kudlicki WA, Bench G, Hoeprich PD, Coleman MA. Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles. Mol Cell Proteomics, 2008, 7(11): 2246-2253
[132] Katzen F. Cell-free protein expression of membrane proteins using nanolipoprotein particles. Biotechniques, 2008, 45: 469
[133] Katzen F, Fletcher JE, Yang JP, Kang D, Peterson TC, Cappuccio JA,Blanchette CD, Sulchek T, Chromy BA, Hoeprich PD, Coleman MA, Kudlicki W.Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach. J Proteome Res, 2008, 7(8): 3535-3542
[2]Singer SJ,Nicolson GL.The fluid mosaic model of the structure of cell membranes.Science,1972,175:720-731
[3]Rothman JE,Lenard J.Membrane asymmetry.Science,1997,195:743-753
[4]隋森芳编著。膜分子生物学。北京:高等教育出版社,2003,P1-5
[5]Bowie JU.Solving the membrane protein folding problem.Nature,2005,438:581-589
[6]Brunecky R,Lee S,Rzepecki PW,Overduin M,Prestwich GD,Kutateladze AG,Kutateladze TG.Investigation of the binding geometry of a peripheral membrane protein.Biochemistry,2005,44(49):16064-16071
[7]Cho W,Stahelin RV.Membrane-protein interactions in cell signaling and membrane trafficking.Annu Rev Biophys Biomol Struct,2005,34:119-151
[8]Gao FP,Cross TA.Recent developments in membrane-protein structural genomics.Genome Biol,2005,6(13):244
[9]Dailey MM,Hait C,Holt PA,Maguire JM,Meier JB,Miller MC,Petraccone L,Trent JO.Structure-based drug design:from nucleic acid to membrane protein targets.Exp Mol Pathol,2009,86(3):141-50
[10]Faller LD.Mechanistic studies of sodium pump.Arch Biochem Biophys,2008,476(1):12-21
[11]Staudinger JL,Lichti K.Cell signaling and nuclear receptors:new opportunities for molecular pharmaceuticals in liver disease.Mol Pharm,2008,5(1):17-34
[12]Wang K,Wong YH.G protein signaling controls the differentiation of multiple cell lineages.Biofactors,2009,35(3):232-8.
[13]Williams C,Hill SJ.GPCR signaling:understanding the pathway to successful drug discovery.Methods Mol Biol,2009,552:39-50
[14] Membrane Protein of Known 3D Structure, [December 2009],http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
[15] Protein Data Bank, [December 2009], http://www.rcsb.org/pdb/home/home.do
[16] Grisshammer R, Tate CG. Overexpression of integral membrane proteins for structural studies. Q Rev Biophys, 1995, 28: 315-422
[17] Ress DC, Chang G, Spencer RH. Crystallographic analyses of ion chennels:Lessons and challenges. J Biol Chem, 2000, 275: 32399-32402
[18] Haines TH. Water transport across biological membranes. FEBS Lett, 1994, 346:115-122
[19] Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science, 1992, 256: 385-387
[20] Agre P, Brown D, Nielsen S. Aquaporin water channels: unanswered questions and unresolved controversies. Curr Opin Cell Biol, 1995, 7: 472-483
[21] Agre P. Aquaporin water channels (Nobel Lecture). Bioscience Rep, 2004, 24(3):127-172
[22] Verkman AS. More than just water channels: unexpected cellular roles of aquaporins. J Cell Sci, 2005, 118(15): 3225-3232
[23] Ishibashi K, Hara S, Kondo S. Aquaporin water channels in mammals. Clin Exp Nephrol, 2009, 13(2): 107-17
[24] Schrier RW. Aquaporin-related disorders of water homeostasis. Drug News Perspect, 2007, 20(7): 447-53
[25] Chepelinsky AB. Structural function of MIP/aquaporin 0 in the eye lens; genetic defects lead to congenital inherited cataracts. Handb Exp Pharmacol, 2009, 190:265-97
[26] Oshio K, Song Y, Verkman AS, Manley GT. Aquaporin-1 deletion reduces osmotic water permeability and cerebrospinal fluid production. Acta Neurochir Suppl,2003, 86: 525-528.
[27] Nishimura H, Yang Y, Lau K, Kuykindoll RJ, Fan Z, Yamaguchi K, Yamamoto T.Aquaporin-2 water channel in developing quail kidney: possible role in programming adult fluid homeostasis. Am J Physiol Regul Integr Comp Physiol, 2007, 293(5): R2147-R2158
[28] Benarroch EE. Aquaporin-4, homeostasis, and neurologic disease. Neurology, 2007, 69(24): 2266-2268
[29] Galamita G, Bishai WR, Preston GM, Guggino WB, Agre P. Molecular cloning and characterization of AqpZ, a water channel from E. coli. J Biol Chem, 1995, 49:29063-29066
[30] Calamita G, Kempf B, Rudd KE, Bonhivers M, Kneip S, Bishai WR, Bremerd E,Agre P. The aquaporin-Z water channel gene of Escherichia coli: Structure,organization and phylogeny. Biol Cell, 1997, 89: 321-329
[31] Calamita G. The Escherichia coli aquaporin-Z water channel. Mol Microbiol,2000, 37(2): 254-262
[32] Borgnia MJ, Kozono D, Calamita G, Maloney PC, Agre P. Functional Reconstitution and Characterization of AqpZ, the E. coli Water Channel Protein. J Mol Biol, 1999,291: 1169-1179
[33] Daniels BV, Jiang JS, Fu D. Crystallization and preliminary crystallograpgic analysis of the Escherichia coli water channel AqpZ. Acta Cryst D, 2004, 60: 561-563
[34] Jiang JS, Daniels BV, Fu D. Crystal structure of AqpZ tetramer reveals two distinct Arg-189 conformations associated with water permeation through the narrowest constriction of the water-conducting channel. J Biol Chem, 2006, 281(1):454-460
[35] Ringler P, Borgnia MJ, Stahlberg H, Maloney PC, Agre P, Engel A. Structure of the water channel AqpZ from Escherichia coli revealed by electron crystallography. J Mol Biol, 1999,291: 1181-1190
[36] Scheuring S, Ringler P, Borgnia M, Stahlberg H, Muller DJ, Agre P, Engel A.High resolution AFM topographs of the Escherichia coli water channel aquaporin Z.EMBO J, 1999, 18(18): 4981-4987
[37] Savage DF, Egea PF, Robles-Colmenares Y, O'Connell JD, Stroud RM. Architecture and selectivity in aquaporins: 2.5 a X-ray structure of aquaporin Z. PLoS Biol, 2003, 1(3): E72
[38] Jensen M, Mouritsen OG. Single-channel water permeabilities of Escherichia coli aquaporins: AqpZ and GlpF. Biophys J, 2006, 90: 2270-2284
[39] Phongphanphanee S, Yoshida N, Hirata F. The statistical-mechanics study for the distribution of water molecules in aquaporin. Chem Phys Lett, 2007,449: 196-201
[40] Wang Y, Schulten K, Tajkhorshid E. What makes an aquaporin a glycerol channel?A comparative study of AqpZ and GlpF. Structure, 2005,13: 1107-1118
[41] Hashido M, Ikeguchi M, Kidera A. Comparative simulations of aquaporin family:AQP1, AQPZ, AQP0 and GlpF. FEBS Lett, 2005, 579: 5549-5552
[42] Pohl P, Saparov SM, Borgnia MJ, Agre P. Highly selective water channel activity measured by voltage clamp: Analysis of planar lipid bilayers reconstituted with purified AqpZ. Proc Natl Acad Sci USA, 2001, 98(17): 9624-9625
[43] Fujiyoshi Y, Mitsuoka K, de Groot BL, Philippsen A, Grubmüller H, Agre P,Engel A. Structure and function of water channels. Curr Opin Struct Biol, 2002, 12(4):509-15
[44] Verkman AS, Mitra AK. Structure and function of aquaporin water channels. Am J Physiol Renal Physiol, 2000, 278(1): F13-F28
[45] Kumar M, Grzelakowski M, Zilles J, Clark M, Meier W. Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z. Proc Natl Acad Sci USA, 2007, 104(52): 20719-20724
[46] Swartz JR. Developing cell-free biology for industrial applications. J Ind Microbiol Biotechnol, 2006, 33: 476-485
[47] Haddoub R, Rutzler M, Robin A, Flitsch SL. Design, synthesis and assaying of potential aquaporin inhibitors. Handb Exp Pharmacol, 2009, 190: 385-402.
[48] Castle NA. Aquaporins as targets for drug discovery. Drug Discov Today, 2005,10(7): 485-93
[49] Yasui M. Molecular mechanisms and drug development in aquaporin water channel diseases: structure and function of aquaporins. J Pharmacol Sci, 2004, 96(3):260-3
[50] Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A,Nielsen S. Aquaporin water channels-from atomic structure to clinical medicine. J Physiol, 2002, 542(1): 3-16
[51] Wang DN, Safferling M, Lemieux MJ, Griffith H, Chen Y, Li XD. Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies. Biochim Biophys Acta, 2003, 1610: 23-36
[52] Tate CG. Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett, 2001, 504(3): 94-98
[53] Wuu JJ, Swartz JR. High yield cell-free production of integral membrane proteins without refolding or detergents. Biochim Biophys Acta, 2008, 1778:1237-1250
[54] Miroux B, Walker JE. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol, 1996,260: 289-298
[55] Arechaga I, Miroux B, Karrasch S, Huijbregts R, Kruijff BD, Runswick MJ,Walker JE. Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F_1F_0 ATP synthase.FEBS Lett, 2000, 482: 215-219
[56] Mohanty AK, Wiener MC. Membrane protein expression and production: effects of polyhistidine tag length and position. Protein Expre Purif, 2004, 33: 311-325
[57] Link JA, Skretas G, Strauch EM, Chari NS, Georgiou G. Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli. Protein Sci, 2008, 17: 1857-1863
[58] Skretas G, Georgiou G. Genetic analysis of G protein-coupled receptor expression in Escherichia coli: Inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor. Biotechnol Bioeng, 2009, 102(2): 357-367
[59] Kiefer H, Vogel R, Maier K. Bacterial expression of G-protein-coupled receptors:prediction of expression levels from sequence. Receptors Channels, 2000, 7: 109-119
[60] Kiefer H, Krieger J, Olszewski JD, Von Heijne G, Prestwich GD, Breer H.Expression of an olfactory receptor in Escherichia coli: purification, reconstitution,and ligand binding. Biochemistry, 1996, 35: 16077-16084
[61] Korepanova A, Moore JD, Nguyen HB, Hua Y, Cross TA, Gao F. Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein. Protein Expr Purif, 2007, 53: 24-30
[62] Chen GQ, Gouaux JE. Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: Efficient regeneration of the fusion protein and selective cleavage with trypsin. Protein Sci, 1996,5:456-467
[63] Pompejus M, Friedrich K, Teufel M, Fritz HJ. High-yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli. Eur J Biochem, 1993,211:27-35
[64] Yao Q, Bevan JL, Weaver RF, Bigelow DJ. Purification of porcine phospholamban expressed in Escherichia coli. Protein Expres Purif, 1996, 8: 463—468
[65] Buck B, Zamoon J, Kirby TL, DeSilva TM, Karim C, Thomas D, Veglia G.Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies. Protein Expres Purif, 2003, 30: 253-261
[66] Arkin IT, Adams PD, MacKenzie KR, Lemmon MA, Brunger AT, Engelman DM.Structural organization of the pentameric transmembrane alpha-helices of phospholamban, a cardiac ion channel. EMBO J, 1994, 13: 4757-4764
[67] Grisshammer R, Duckworth R, Henderson R. Expression of a rat neurotensin receptor in Escherichia coli. Biochem J, 1993, 295: 571-576
[68] Tucker J, Grisshammer R. Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem J, 1996, 317: 891-899
[69] Stanasila L, Massotte D, Kieffer BL, Pattus F. Expression of δ, μ and κ human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins. Eur J Biochem, 1999, 260: 430-438
[70] Lemmon MA, Flanagan JM, Hunt JF, Adair BD, Bormann BJ, Dempsey CE,Engelman DM. Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices. J Biol Chem, 1992, 267: 7683-7689
[71] Ramachandran S, Lu HL, Prabhu U, Ruoho AE. Purification and characterization of the guinea pig sigma-1 receptor functionally expressed in Escherichia coli. Protein Expr Purif, 2007, 51: 283-292
[72] Weiss HM, Grisshammer R. Purification and characterization of the human adenosine A_(2a) receptor functionally expressed in Escherichia coli. Eur J Biochem,2002, 269: 82-92
[73] Roosild TP, Greenwald J, Vega M, Castronovo S, Riek R, Choe S. NMR structure of Mistic, a membrane-integrating protein for membrane protein expression.Science, 2005, 307: 1317-1321
[74] Kulothungan SR, Das M, Johnson M, Ganesh C, Varadarajan R. Effect of crowding agents, signal peptide, and chaperone SecB on the folding and aggregation of E. coli maltose binding protein. Langmuir, 2009,25(12): 6637-48.
[75] Katzen F, Chang G, Kudlicki W. The past, present and future of cell-free protein synthesis. Trends Biotechol, 2005, 23: 150-156
[76] Klammt C, Schwarz D, Lohr F, Schneider B, Dotsch V, Bernhard F. Cell-free expression as an emerging technique for the large scale production of integral membrane protein. FEBS J, 2006, 273: 4141-4153
[77] Schwarz D, Klammt C, Koglin A, Lohr F, Schneider B, Dotsch V, Bernhard F.Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies. Methods, 2007, 41: 355-369
[78] Liguori L, Marques B, Villegas-Mendez A, Rothe R, Lenormand JL. Production of membrane proteins using cell-free expression systems. Expert Rev Proteomics,2007, 4(1): 79-90
[79] Endo Y, Sawasaki T. Cell-free expression systems for eukaryotic protein production. Curr Opin Biotechnol, 2006, 17: 373-380
[80] Yin G, Swartz JR. Enhancing multiple disulfide bonded protein folding in a cell-free system. Biotechnol Bioeng, 2004, 86:188-195
[81] Goerke AR, Swartz JR. Development of cell-free protein synthesis platforms for disulfide bonded proteins. Biotechnol Bioeng, 2008, 99: 351-367
[82] Klammt C, Lohr F, Schafer B, Haase W, Dotsch V, Ruterjans GC, Bernhard F.High level cell-free expression and specific labeling of integral membrane proteins.Eur J Biochem, 2004, 271:568-580
[83] Goerke AR, Swartz JR. High-level cell-free synthesis yields of proteins containing site-specific non-natural amino acids. Biotechnol Bioeng, 2009, 102(2):400-416
[84] Rungpragayphan S, Nakano H, Yamane T. PCR-linked in vitro expression: a novel system for high-throughput construction and screening of protein libraries.FEBS Lett, 2003, 540: 147-150
[85] Keller T, Schwarz D, Bernhard F, Dotsch V, Hunte C, Gorboulev V, Koepsell H.Cell free expression and functional reconstitution of eukaryotic drug transporters.Biochemistry, 2008,47: 4552-4564
[86] Shimada Y, Wang ZY, Mochizuki Y, Kobayashi M, Nozawa T. Functional expression and characterization of a bacterial light-harvesting membrane protein in Escherichia coli and cell free synthesis system. Biosci Biotechnol Biochem, 2004,68(9): 1942-1948
[87] Klammt C, Schwarz D, Fendler K, Haase W, Dotsch V, Bernhard F. Evaluation of detergents for the soluble expression of a-helical and β-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system. FEBS J, 2005,272: 6024-6038
[88] Elbaz Y, Steiner-Mordoch S, Danieli T, Schuldiner S. In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state. Proc Natl Acad Sci USA, 2004, 101: 1519-1524
[89] Berrier C, Park KH, Abes S, Bibonne A, Betton JM, Ghazi A. Cell free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent. Biochemistry, 2004, 43: 12585-12591
[90] Klammt C, Schwarz D, Eifler N, Engel A, Piehler J, Haase W, Hahn S, Dotsch V,Bernhard F. Cell-free production of G protein-coupled receptors for functional and structural studies. J Struct Biol, 2007, 158: 482-493
[91] Gourdon P, Alfredsson A, Pedersen A, Malmerberg E, Nyblom M, Widell M,Berntsson R, Pinhassi J, Braiman M, Hansson O, Bonander N, Karlsson G, Neutze R. Optimized in vitro and in vivo expression of proteorhodopsin: A seven-transmembrane proton pump. Protein Expr Purif, 2008, 58: 103-113
[92] Martin M, Albanesi D, Alzari M, Mendoza D. Functional in vitro assembly of the integral membrane bacterial thermosensor DesK. Protein Expr Purif, 2009, 66: 39-45
[93] Ishihara G, Goto M, Saeki M, Ito K, Hori T, Kigawa T, Shirouzu M, Yokoyama S.Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin fusion vectors. Protein Expr Purif, 2005,41: 27-37
[94] Liguori L, Marques B, Lenormand JL. A bacterial cell-free expression system to produce membrane proteins and proteoliposomes: from cDNA to functional assay.Curr Protoc Protein Sci, 2008, 5.22.1-5.22.30
[95] Liguori Lavini, Blesneac I, Madern D, Vivaudou M, Lenormand JL. Single-step production of functional OEP24 proteoliposomes. Protein Expr Purif, 2010, 69:106-111
[96] Marques B, Liguori L, Paclet M-H, Villegas-Mendez A, Rothe R, Morel F,Lenormand JL. Liposome-Mediated Cellular Delivery of Active gp91~(phox). PLoS ONE,2007,2(9): e856
[97] Shim JW, Yang MM, Gu LQ. In vitro synthesis, tetramerization and single channel characterization of virus-encoded potassium channel Kcv. FEBS Lett, 2007,581: 1027-1034
[98] van Dalena A, van der Laanb M, Driessenb A, Killiana JA, de Kruijff B.Components required for membrane assembly of newly synthesized K~+ channel KcsA.FEBS Lett, 2002, 511: 51-58
[99] Kalmbach R, Chizhov I, Schumacher MC, Friedrich T, Bamberg E, Engelhard M.Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes. J Mol Biol, 2007, 371: 639-648
[100] Nagamori S, Vazquez-Ibar JL, Weinglass AB, Kaback HR. In vitro synthesis of lactose permease to probe the mechanism of membrane insertion and folding. J Biol Chem, 2003, 278: 14820-14826
[101] Liguori L, Marques B, Villegas-Mendez A, Rothe R, Lenormand JL.Liposomes-mediated delivery of pro-apoptotic therapeutic membrane proteins. J Control Release, 2008, 126: 217-227
[102] Park KH, Berrier C, Lebaupain F, Pucci B, Popot JL, Ghazi A, Zito F.Flurinated and hemiflurinated surfactants as alternatives to detergents for membrane protein cell-free synthesis.Biochem J,2007,403:183-187
[103]Schnaitman CA.Protein Composition of the cell wall and cytoplasmic membrane of Escherichia coli.J Bacteriol,1970,104(2):890-901
[104]Peker B,Wuu JJ,Swartz JR.Affinity purification of lipid vesicles.Biotechnol Prog,2004,20:262-268
[105]Sambrook J,Fritsch E F,Maniatis T.分子克隆实验指南(第三版),北京:科学出版社,2002,P26-99
[106]Coligan JE,Dunn BM,Speicher DW,Wingfield PT.精编蛋白质科学实验指南,北京:科学出版社,2007,P291-334
[107]LaVallie ER,DiBlasio EA,Kovacic S,Grant KL,Schendel PF,McCoy JM.A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E.coli cytoplasm.Biotechnology,1993,11:187-193
[108]Zhang Y,Olsen DR,Nguyen KB,Olson PS,Rhodes ET,Mascarenhas D.Expression of eukaryotic proteins in soluble form in Escherichia coli.Protein Expres Purif,1998,12:159-165
[109]Nygren PA,Stahl S,Uhlen M.Engineering proteins to facilitate bioprocessing.Trends Biotechnol,1994,12:184-188
[110]Samuelsson E,Moks T,Nilsson B,Uhlen M.Enhanced in vitro refolding of insulin-like growth factor I using a solubilizing fusion partner.Biochemistry,1994,33:4207-4211
[111]Power RF,Conneely OM,McDonnell DP,Clark JH,Butt TR,Schrader WT,O'Malley BW.High level expression of a truncated chicken progesterone receptor in Escherichia coli.J Biol Chem,1990,265:1419-1424
[112]Pryor KD,Leiting B.High-level expression of soluble protein in Escherichia coli using a His6-tag and maltose-binding protein double-affinity fusion system.Protein Expres Purif,1997,10:309-319
[113]Peng L,Xu ZN,Fang XM,Wang F,Cen PL.High-level expression of soluble human Beta-Defensin-2 in E.coli.Process Biochem,2004,39:2199-2205
[114]Xu ZN,Zhong ZX,Huang L,Peng L,Wang F,Cen PL.High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression. Appl Microbiol Biotechnol, 2006, 72: 471-479
[115] Zhong ZX, Xu ZN, Peng L, Huang L, Fang XM, Cen PL. Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli.Appl Microbiol Biotechnol, 2006, 71(5): 661-667
[116] Kozono D, Ding X, Iwasaki I, Meng X, Kamagata Y, Agre P, Kitagawa Y.Functional expression and characterization of an archaeal aquaporin: AqpM from Methanothermobactermarburgensis. J Biol Chem, 2003,278(12): 10649-10656
[117] Hiroaki Y, Tani K, Kamegawa A, Gyobu N, Nishikawa K, Suzuki H, Walz T,Sasaki S, Mitsuoka K, Kimura K, Mizoguchi A, Fujiyoshi Y. Implications of the aquaporin-4 structure on array formation and cell adhesion. J Mol Biol, 2006, 355(4):628-639
[118] Karlsson M, Fotiadis D, Sjovall S, Johansson I, Hedfalk K, Engel A, Kjellbom P.Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris. FEBS Lett, 2003, 537(1-3): 68-72
[119] Hall MN, Gabay J, Debarbouille M, Schwartz M. A role for mRNA secondary structure in the control of translation initiation. Nature, 1982, 295: 616-618
[120] Brodsky LI, Ivanov VV, Kalaydzidis YL, Leontovich AM, Nikolaev VK,Feranchuk SI, Drachev VA. GeneBee-NET: Internet-based server for analyzing biopolymers structure. Biochemistry, 1995, 60: 923-928
[121] DeGrip WJ. Thermal stability of rhodopsin and opsin in some novel detergents.Methods Enzymol, 1982, 81: 256-265
[122] Odahara T. Stability and solubility of integral membrane proteins from photosynthetic bacteria solubilized in different detergents. Biochim Biophys Acta,2004, 1660: 80-92
[123] Ganoza MC, Kofoid EC, Marliere P, Louis BG. Potential secondary structure at translation-initiation sites. Nucleic Acids Res, 1987, 15(1): 345-360
[124] de Smit MH, van Duin J. Secondary structure of the ribosome binding site determines translational efficiency: A quantitative analysis. Proc Natl Acad Sci USA,1990, 87: 7668-7672
[125] Kimura S, Iyanari T. High-level expression of porcine liver cytochrome P-450 reductase catalytic domain in Escherichia coli by modulating the predicted local secondary structure of mRNA. J Biochem, 2003, 134: 403-413
[126] Son JM, Ahn JH, Hwang MY, Park CG, Choi CY, Kim DM. Enhancing the efficiency of cell-free protein synthesis through the polymerase-chain-reaction-based addition of a translation enhancer sequence and the in situ removal of the extra amino acid residues. Anal Biochem, 2006, 351: 187-192
[127] Kimura S, Umemura T, Iyanagi T. Two-cistronic expression plasmids for high-level gene expression in Escherichia coli preventing translational initiation inhibition caused by the intramolecular local secondary structure of mRNA. J Biochem, 2005, 137: 523-533
[128] Tschantz WR, Paetze M, Cao GQ, Suciu D, Inouye M, Dalbey RE. Characterization of a soluble, catalytically active form of Escherichia coli leader peptidase: requirement of detergent or phospholipid for optimal activity. Biochemistry 1995,34:3935-3941
[129] Choo KH, Tan TW, Ranganathan S. SPdb - a signal peptide database. BMC Bioinformatics, 2005, 6: 249
[130] Katzen F, Peterson TC, Kudlicki W. Membrane protein expression: no cells required. Trends Biotechnol, 2009, 27(8): 455-460
[131] Cappuccio JA, Blanchette CD, Sulchek TA, Arroyo ES, Kralj JM, Hinz AK,Kuhn EA, Chromy BA, Segelke BW, Rothschild KJ, Fletcher JE, Katzen F, Peterson TC, Kudlicki WA, Bench G, Hoeprich PD, Coleman MA. Cell-free co-expression of functional membrane proteins and apolipoprotein, forming soluble nanolipoprotein particles. Mol Cell Proteomics, 2008, 7(11): 2246-2253
[132] Katzen F. Cell-free protein expression of membrane proteins using nanolipoprotein particles. Biotechniques, 2008, 45: 469
[133] Katzen F, Fletcher JE, Yang JP, Kang D, Peterson TC, Cappuccio JA,Blanchette CD, Sulchek T, Chromy BA, Hoeprich PD, Coleman MA, Kudlicki W.Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach. J Proteome Res, 2008, 7(8): 3535-3542